1. Field of the Invention
This present invention is directed to cell culture and, more particularly, to a three-dimensional culture and harvest system for anchorage-dependent cells.
2. Description of Related Art
There has recently been a resurgence of interest in stem cell research. Stem cells are multi-potent and plastic, which enables them to be induced to differentiate into various cell types. Almeida-Porada et al., “Adult Stem Cell Plasticity and Methods of Detection,” Rev Clin Exp Hematol 5(1), 26-41 (2001). Adult stem (AS) cells are preferable in most applications not only because the cell sources are less controversial and more readily available than embryonic stem (ES) cells, but also the adult stem cells are more easily induced into the final differentiated cell types needed in applications. Clarke, D. et al, “Differentiation Potential of Adult Stem Cells,” Curr Opin Genet Dev 11(5), 575-580 (2001). To date, AS cells are not considered suitable substitutes for ES cells because of the differences in their proliferation capacity in vitro. Gage, F. H., “Mammalian Neural Stem Cells,” Science 287 (5457), 1433-1438 (2000); NIH bioethics guideline. ES cells (especially in murine models) have essentially unlimited proliferation capacity in vitro, which means that they can be expanded greatly for applications. On the other hand, most AS cells can only proliferate for about 5-12 passages in vitro and then stop proliferating, but randomly differentiate into other cell types. Pittenger, M. F. et al., “Multilineage Potential of Adult Human Mesenchymal Stem Cells,”Science 284 (5411), 143-147 (1999). This characteristic has heretofore severely limited the usefulness of AS cells in therapeutic applications.
Most mammalian cells are anchorage-dependent (except cells in fluid, such as lymphatic and hematopoietic cells). In vitro culture of the anchorage-dependent cells traditionally has been limed by two primary factors. One is the limited space of the culture vessels. Cells will only proliferate exponentially in log phase when there is adequate room in the culture vessels. Once the cells establish contact with each other, their proliferation slows down and eventually stops or the cells even signal each other to die by apoptosis. The other limiting factor is the suitability of substrates for cell attachment. Inappropriate attachment often alters the cellular functions and all the observed structures and functions thereafter. Both factors cause potential artifacts that can affect the outcome of cell biological studies or cell-transplantation or tissue engineering applications.
Several approaches have been used to avoid contact inhibition of cell proliferation by providing sufficient space or surface on which the cells can grow. One approach has been to split cells by protease treatment to detach the cells from the culture surface before the cell-density becomes confluent. Trypsin is commonly used but others, such as collagenase, are also used in some circumstances. Protease-related cells typically are rounded up, collected, pelleted by centrifugation, re-suspended in fresh culture media, and distributed in multiple culture vessels. Each new vessel contains a fraction of the original cell number, and thus has sufficient new surface to allow cell proliferation until confluence again occurs. Such a splitting process can be repeated several times, until the cells stop proliferating due to other reasons.
Another approach has been to increase the time before successive cell splitting by lowering the minimum number of cells to be seeded into vessels or increasing the maximum number of cells that the culture vessel can accommodate before confluence. Because cells naturally prefer to be in the vicinity of other cells to support each other, the minimum number that can be seeded initially in culture vessel is often limited by the support rendered by cell—cell interaction, communication and the release of growth factors and the like. Therefore, enriched culture media have been used to support cell growth and proliferation, allowing lower minimum cell number to be seeded in the vessel, and allowing a longer period before the cells have to be split again.
Another way of increasing the time of culture between each split has been to grow cells in three-dimensional matrix. A three-dimensional matrix typically has a thick layer of matrix such as collagen on the surface of culture vessel, or microspheres of concentrated matrix. In such a three-dimensional matrix, cells can grow into multiple layers in 3 dimensions, thereby permitting a longer culture period before confluence. To modulate cell attachment to a substrate, various natural and synthetic substrates have been developed such as those involving short-peptides and sugar-motifs and the like.
Essentially all these approaches involve splitting cells with disruptive techniques such as proteases, cold treatment, or EDTA treatment combined with cell scraping to detach the cells from the substrate (either charged surfaces or three-dimensional matrix). Either of these splitting techniques can have detrimental effects on cell structure and function. For example, proteases digest away cell surface molecules that are essential not only for cell attachment to substrates but also for signaling molecules that are important for cell function. Cold treatment has been shown to adversely affect various cellular processes such as cytoskeleton arrangement, membrane trafficking pathways, and the like. EDTA treatment and mechanical scraping has been shown to adversely affect cell functions because the cells round up and are prone to damage by mechanical scraping. In the case of three-dimensional culture in matrix, protease treatment is the only way presently known to split cells. For cell transplantation or cell seeding onto tissue-engineered scaffolds, cells with temporarily damaged functions (due to disruptive cell splitting techniques) are currently being used because the current culture methods cannot offer a better alternative.